Bacillus anthracis is the causative agent of anthrax, a zoonotic disease that is transmitted by uptake of infectious spores. Germination of spores and massive expansion of the resulting vegetative forms precipitate the rapidly lethal disease anthrax, which affects most mammals including humans. B. anthracis has served as a model organism for infectious diseases and was used to first demonstrate that bacteria represent the causative agents of infectious diseases. Further, live-attenuated spore vaccines (LSVs) were used for the first demonstration that infected hosts can mount immune responses protective against wild-type B. anthracis. One of these LSVs, B. anthracis strain Sterne (pXO1+, pXO2-) lacks the pXO2 plasmid responsible for expression of the poly-D-?-glutamic acid capsule (PDGA) and is used globally to prevent anthrax disease in domesticated animals. Importantly, the correlate of LSV-induced anthrax protective immunity is not yet known, however it is appreciated that LSV-derived opsonophagocytic antibodies promote clearance of the pathogen and sterilizing immunity. In the United States, AVA (BioThraxTM), a precipitate of Sterne-type culture supernatants adsorbed to aluminum hydroxide, is used as the anthrax vaccine for humans. AVA provides only partial protection in several animal models and the vaccine does not confer sterilizing immunity. AVA's correlate for protection, antibodies against the lethal toxi component protective antigen (PagA), neutralize the toxin but cannot promote opsonophagocytic clearance of the pathogen. Here we seek to address a key unanswered question for the field of bacterial pathogenesis: what is the correlate of protective immunity derived from LSVs? Obviously, the PDGA capsule can be ruled out, because the Sterne strain does not elaborate capsule. Our current appreciation of the envelope of vegetative forms implicates two main envelope structures as candidates, the secondary cell wall polysaccharide (SCWP) and the S-layer. To evaluate the contributions of the SCWP and the S-layer as protective antigens towards anthrax pathogenesis and protective immunity requires the molecular genetic analysis of the pathogen. We have shown that the SLH domain of 2 S-layer proteins (Sap and EA1) and 22 S-layer associated proteins (BslA-T) binds to the SCWP when it is pyruvylated via the csaB gene product and linked to the peptidoglycan of B. anthracis. Unliked pagA, both csaB and bslA are required for anthrax pathogenesis, however the genes that enable synthesis and assembly of the SCWP and its associated S-layer are not yet known. The knowledge gap is addressed in this proposal, which will explore the biochemical underpinnings of ordered S-layer and S-layer associated protein assembly on the SCWP.